Treatment of Cognitive Disorders with (R)-7-Chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-Carboxamide and Pharmaceutically Acceptable Salts Thereof

ABSTRACT

(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide has been found to have procognitive effects in humans at unexpectedly low doses. Thus, (R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide and pharmaceutically acceptable salts thereof can be used at unexpectedly low doses to improve cognition.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/129,782, filed Aug. 9, 2011, now U.S. Pat. No. 8,642,638 granted Feb.4, 2014, which is a National Phase application of InternationalApplication No. PCT/US2009/065173, filed Nov. 19, 2009, which designatedthe United States and was published in English, and which further claimsthe benefit of priority from U.S. Provisional Application No.61/116,106, filed Nov. 19, 2008. The foregoing related applications, intheir entirety, are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Nicotinic acetylcholine receptors (nAChR) form a family of ion channelsactivated by acetylcholine. Functional receptors contain five subunitsand there are numerous receptor subtypes. Studies have shown thatcentral nicotinic acetylcholine receptors are involved in learning andmemory. Nicotinic acetylcholine receptors of the alpha7 subtype areprevalent in the hippocampus and cerebral cortex.

WO 2003/055878 describes a variety of agonists of the alpha7 nAChR saidto be useful for improving cognition. WO 2003/055878 suggests thatcertain agonists of the alpha7 nAChR are useful for improvingperception, concentration, learning or memory, especially aftercognitive impairments like those occurring for example insituations/diseases/syndromes such as mild cognitive impairment,age-associated learning and memory impairments, age-associated memoryloss, Alzheimer's disease, schizophrenia and certain other cognitivedisorders. Among the compounds described are(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide andpharmaceutically acceptable salts thereof.

SUMMARY OF THE INVENTION

It has been found that(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide canhave procognitive effects in humans at unexpectedly low doses. Thus,(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide andpharmaceutically acceptable salts thereof can be used at unexpectedlylow doses to improve cognition in individuals suffering from impairedcognition and in healthy individuals (i.e., individuals that are notsuffering from an apparent cognitive deficit). For example, it can beused to improve cognition in patients suffering from Alzheimer'sdisease, schizophrenia and other disorders such as otherneurodegenerative diseases (e.g., Huntington's disease or Parkinson'sdisease) and attention deficit disorder. It can be used treat certaindisorders, e.g., Alzheimer's disease, schizophrenia (e.g., paranoidtype, disorganized type, catatonic type, and undifferentiated type),schizophreniform disorder, schizoaffective disorder, delusionaldisorder, positive symptoms of schizophrenia, negative symptoms ofschizophrenia at a daily dose of 3 mg, 2.70 mg, 2.50 mg, 2.25 mg, 2 mg,1.75 mg, 1.50 mg, 1.25 mg, 1 mg, 0.7, 0.5, 0.3 mg or even 0.1 mg. Thecompound can be used to improve one or more aspects of cognition, e.g.,one or more of: executive function, memory (e.g., working memory),social cognition, visual learning, verbal learning and speed ofprocessing.

Described herein are methods for treating a patient by administering apharmaceutical composition that comprises(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide orpharmaceutically acceptable salt thereof at a daily dose of: 3 mg, 2.70mg, 2.50 mg, 2.25 mg, 2 mg, 1.75 mg, 1.50 mg, 1.25 mg, 1 mg, 0.7 mg, 0.5mg, 0.3 mg, or 0.1 mg. The treatment can improve one or more facets ofcognition (e.g., visual motor skill, learning, delayed memory,attention, working memory, visual learning, speed of processing,vigilance, verbal learning, visual motor function, social cognition,long term memory, executive function, etc.). The methods can be used totreat: Alzheimer's disease, schizophrenia (e.g., paranoid type,disorganized type, catatonic type, and undifferentiated type),schizophreniform disorder, schizoaffective disorder, delusionaldisorder, positive symptoms of schizophrenia or negative symptoms ofschizophrema.

“Dose” is the amount of active pharmaceutical ingredient (API)administered to a patient. For example, 1 mg dose means 1 mg of API wasadministered to each patient each day.

“Active Pharmaceutical Ingredient” is defined as either(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamidehydrochloride,(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide,(R)-7-chloro-N(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamidehydrochloride monohydrate or(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamidehydrochloride solvate.

Where solvate represents a stoichiometric ratio of 0.1 to 10 moleculesof solvent compared to(R)-7-chloro-N(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamidehydrochloride or(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide.Solvent molecules include but are not limited to water, methanol, 1,4dioxane, ethanol, isopropanol or acetone. In some cases, water is thepreferred solvate.

“The test compound” is defined as(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamidehydrochloride.

“EC_(ref)” is the concentration of drug which elicits equal response inoocytes transfected with cloned human alpha7 receptor at 50 μMacetylcholine. Maximum stimulation of the cloned human alpha7 receptoroccurs at a concentration of >250 μM of acetylcholine.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 depicts the results of a study on the effect of the test compoundon P50 gating ratio. Specifically, FIG. 1A depicts the baseline-adjustedaverage P50 gating ratio (T/C) as a function of group assignment[F=1.16, P=0.36]. The standard errors of each mean are noted in thelegend. The bars (left to right) represent placebo, 0.3 mg testcompound, and 1.0 mg test compound. FIG. 1B depicts thebaseline-adjusted average P50 difference (C-T) as a function of groupassignment [F=3.97, P=0.07]. The standard errors of each mean are notedin the legend. The bars left to right represent placebo, 0.3 mg testcompound, and 1.0 mg test compound.

FIG. 2 depicts the results of a study on the effect of the test compoundon N100 gating ratio. Specifically, FIG. 2A depicts baseline-adjustedaverage N100 gating ratio (T/C) as a function of group assignment[F=3.04, P=0.10]. The standard errors of each mean are noted in thelegend. The bars left to right represent placebo, 0.3 mg test compound,and 1.0 mg test compound. FIG. 2B depicts baseline-adjusted average N100amplitude difference (C-T) as a function of group assignment [F=1.02,P=0.38]. The standard errors of each mean are noted in the legend. Thebars left to right represent placebo, 0.3 mg test compound, and 1.0 mgtest compound.

FIG. 3 depicts the results of a, study on the effect of the testcompound on MMN amplitude and P300 amplitude. Specifically, FIG. 3Adepicts MMN as a function of group assignment [F=4.96, P=0.02]. Thestandard errors of each mean are noted in the legend. The bars left toright represent placebo, 0.3 mg test compound, and 1.0 mg test compound.FIG. 3B depicts P300 amplitude (in microvolts relative to prestimulusvoltage) measured at Pz scalp in response evoked by a rare butunattended stimulus. Group assignment effect: F=6.88, P=0.008. Thestandard errors of each mean are noted in the legend. The bars left toright represent placebo, 0.3 mg test compound, and 1.0 mg test compound.

DETAILED DESCRIPTION OF THE INVENTION

Described below are human clinical trials demonstrating that(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide elicitspositive effects on cognition at an unexpectedly low daily dose of 1 mgor less. The positive effects are observed in both patients sufferingfrom schizophrenia and in normal subjects. Also described below arestudies showing that the free concentration of(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide inhumans administered at daily 1 mg dose (of(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamidehydrochloride) is at least an order of magnitude lower than thatexpected to be required to exert a positive effect on cognitivefunction, or can improve sensory electrophysiological responses whichcorrelate with improved cognitive and functional performance inschizophrenia patients. Also described below are studies demonstratingthat that(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide has anunexpectedly long half-life in humans compared to that expected based onpre-clinical studies in animals.

Because (R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamidecan improve cognition at an unexpectedly low free plasma concentration,it is less likely to elicit harmful side-effects on its own and is lesslikely to exhibit harmful interactions with other drugs. Due to theunexpectedly low free plasma concentration required and the longhalf-life,(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide isexpected to have special drug properties. These properties include ahigh margin of safety and a favorable dosing regimen (e.g., once dailydosing), both of which are highly advantageous for treating patientswith cognitive defects as well as patients that are required to takeadditional medications.

Effect on Cognition in Schizophrenia Patients

The studies described below demonstrate that(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamidehydrochloride can improve sensory electrophysiological responses whichcorrelate with improved cognitive and functional performance inschizophrenia patients. These effects were observed at a daily dose aslow as 0.3 mg.

Impairment of the ability of the central nervous system to inhibitirrelevant sensory information has long been used as a model forunderstanding the deficits of attention seen in schizophrenic patients.Two approaches to the measurement of this ability have commonly beenemployed [see (Heinrichs, 2004; Potter et al., 2006; Turetsky et al.,2007; Umbricht and Krljes, 2005) for reviews and meta-analyses]: (1) thesensory gating paradigm in which the presentation of one stimulusnormally suppresses the response elicited by a stimulus which rapidlyfollows it while schizophrenic patients typically exhibit lesssuppression (gating) of the second response; and (2) the oddball ororienting paradigm in which a rare or unexpected event elicits adiminished response in schizophrenic patients because attentionalresources are inappropriately focused on less salient aspects of theenvironment.

Two responses are commonly used to assess brain activity: (1) theauditory P50 response elicited by the second member of a pair of clicks;and (2) the mismatch negativity (MMN) or N2 response evoked by a rarelyoccurring pure tone of no instructed relevance to the patient.Abnormalities in both P50 gating and the MMN have been reported inschizophrenic patients. Described below are studies assessing both ofthese responses in patients treated with(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamidehydrochloride salt (“the test compound”). Also presented below arestudies assessing the influence of the test compound on the N100 andP300 components of the evoked response. These components emerge afterthe P50 component and are as much related to attention to, and memoryfor, task relevant stimuli as to the neural processes by which taskirrelevant stimuli are filtered (Turetsky et al., 2007; and Sandman andPatterson, 2000).

The neurobiology of P50 sensory gating is well documented in studies ofhuman and animal subjects. Its regulation relies heavily on theintegrity of the hippocampus and pathways that provide input to thehippocampus (Adler et al., 1998). For example, lesions of thecholinergic pathway originating in the medial septal nucleus disrupt thegating response, as do antagonists of low affinity nicotinic receptors.Cholinergic agonists, including nicotine itself (Adler et al., 1993;Duncan et al., 2001), have been shown to enhance P50 gating (Freedman etal., 2001; Olincy et al., 2006).

The neurobiology of the MMN is more complex. Imaging studies suggestthat the primary and secondary auditory cortices in the temporal lobeare important for its generation (Naatanen and Alho, 1995). Thedorsolateral prefrontal cortex also contributes (Schall et al., 2003).The neurotransmitter systems underlying the MMN are understudied andlargely unknown. Yet, as is the case for P50, nicotinic cholinergicsystems appear important (Baldeweg et al., 2006; Dunbar et al., 2007).

The sensitivity of P300 and N100 to cholinergic compounds has been knownfor many years (Dierks et al., 1994; Kaga et al., 1992). Variouscholinergic antagonists—such as scopolamine—profoundly reduce theamplitudes of these components. In contrast, the components are markedlyimproved in amplitude by cholinesterase inhibitors (Katada et al., 2003;Werber et al., 2001) and other compounds that enhance cholinergicactivity (Easton and Bauer, 1997).

The test described above was used to study the effect of the testcompound on cognition in patients suffering from schizophrenia. Prior totesting, the patients were dosed with: 1 mg of the test compound daily,0.3 mg of the test compound daily or were administered a placebo for 20days. Subjects were tested as described below.

P50 waves were elicited by clicks, 1 msec in duration, grouped in pairsin the classic S1-S2 sequence and presented through earpieces insertedinto the auditory canals. Click intensity was adjusted individually to50 dB above the hearing threshold. The offset-to-onset time from S1 toS2 was fixed at 500 msec. The offset-to-onset time between click pairswas varied from 7-11 sec. A total of 30 pairs of clicks were presentedduring each of 5 or more trial blocks with a one minute rest periodinterposed between each block.

EEG responses to the clicks were amplified to a gain of 10K and filtered(bandpass=3-30 Hz, 12 db roll-off). They were collected from 63 tinelectrodes positioned by an electrode cap (Compumedics Neuroscan, Inc.).Additional electrodes of the same type were applied to the mid-forehead(ground) and in a vertical orientation above and below the left eye.Interelectrode impedances were maintained below 10 kOhms. All recordingswere made with the subject sitting upright and relaxed but awake.

The EEG and eye movement signals were sampled by an analog-to-converterprogrammed to retain EEG activity from 50 msec preceding to 325 msecfollowing click onset. The sampling rate was 1000 Hz. The digitizedsignals were stored in a database for subsequent analysis.

The 150 sweeps of S1 and S2 responses were screened and sweeps withvoltage deviations greater than 100 microvolts in the eye movementchannels were rejected. The remaining accepted sweeps were formed intotime point averages. While blinded to group assignment, the investigatorvisually examined the evoked potential waveforms at the FCz electrodesite. When possible, the investigator identified a negative troughimmediately prior to the P50, the P50 itself, and the following N100component. Admittedly, a distinct P50 component could not be visuallyidentified in all patients at all time points. In those cases, the datawas coded as missing.

P50 response amplitude was calculated as the voltage difference betweenthe P50 peak and the preceding negative trough. The P50 gating ratio wasthen calculated after (Olincy et al., 2006) as the amplitude of the P50response to the second (test) stimulus divided by the amplitude of theP50 response to the first (conditioning) stimulus. A small gating ratiois considered normal or optimal. The P50 amplitude difference (Fuerst etal., 2007) was also measured. It was the amplitude of the conditioningstimulus P50 response minus the amplitude of the test stimulus P50response. A large P50 amplitude difference indicates normal gating.

N100 amplitude was calculated as the peak voltage of N100 minus theaverage voltage during the brief, 50 msec prestimulus period. As was thecase for P50, N100 responses to the conditioning and test stimuli werecalculated as ratios as well as differences.

The MMN and P300 components were elicited during the so-called oddballsequence. The stimulus sequence was a series of lower (500 Hz) andhigher (1000 Hz) pitched pure tones presented at a rate of 1 tone per0.6 sec. The tones were 50 msec in duration, 50 dB above hearing level,and randomly interspersed. The higher pitched tone was the oddballevent. Across the series of 600 tones, it occurred at a probability of0.2. The other tone occurred at the complementary probability of 0.8.Patients were instructed to ignore the tones and instead attend to amagazine held in the lap.

During the task, EEG and EOG activity were digitized at a rate of 500 Hzper channel for 50 msec preceding and 500 msec following stimulus onset.Trials contaminated by eyeblinks or eye movements were removed. Anoff-line program digitally filtered (bandpass=0.1-30 Hz, 12 db roll-off)responses to the rare and frequent events and constructed averaged eventrelated responses for each electrode. At the FCz electrode, the MMN wasmeasured by an automated algorithm that computed the summed amplitude,relative to the prestimulus baseline, over a 100-200 msec time windowfollowing the onsets of the rare (oddball) and frequent tones. MMN wasthen recalculated as the voltage difference between these responses.P300 amplitude was measured at the Pz electrode site as the peakamplitude between 250 and 500 msec following stimulus onset.

The plan for the analysis of the EEG measures was developed prior tobreaking of the blind. It was based on the study design involving 3groups (n=8 high dose, n=8 moderate dose, n=4 placebo) and 4 time points(1 predrug+3 postdrug). The plan offered several alternative strategiesbased upon the completeness and quality of the recordings.Unfortunately, in the case of the P50/N100 gating study, it wasnecessary to discard several patients and post-treatment assignment timepoints from the analysis because, in those instances, a P50 waveform wasnot identifiable and therefore could not be measured. This problem hasbeen acknowledged in the literature, but has not been discussed asopenly and frequently as a skeptical scientist would like. For theanalysis of P50 and N100, we adopted strategy 1 b: “If many postdrugdata points are missing/corrupted, then the remaining postdrug datapoints will be averaged together to create a single postdrug datapoint.” The significant number of missing or unmeasurable P50's,unfortunately, removed another of our analysis options, wherein we hopedto focus on the subgroup of patients who showed the poorest sensorygating at baseline and might show the strongest improvement in gatingafter treatment. Of the 12 patients who provided valid and measurableP50 responses, 2 were in the placebo group, and 5 were in each of thetwo active dose groups.

FIGS. 1A and 1B present the results of simple analyses of covariancewherein all time points during the treatment period with valid data wereaveraged together to yield a single value. This value was then adjustedby regressing it against the baseline value and estimating a new valueas if all patients possessed the same baseline. Then, a simple F testwas performed. In support of the assumption of no significantdifferences between the treatment groups at the baseline (i.e., beforetreatment), we conducted simple ANOVAs evaluating the effect oftreatment on all of the evoked potential components discussed presently.In no case did treatment significantly affect the baseline value. FIG.1A shows a non-significant [F=1.16, P=0.36] reduction (i.e.,normalization) of the P50 gating ratio among patients receiving the 1.0mg dose of the test compound. In contrast, FIG. 1B shows the P50amplitude difference score—a metric with superior reliability. Itlikewise shows normalization at the high dose. However, in this case,the change approaches statistical significance [F=3.97, P=0.07].

FIGS. 2A and 2B present an identical analysis of the N100 gating ratioand amplitude difference. Here, the gating ratio demonstrates a morereliable effect of the medication [F=3.04, P=0.10] than does theamplitude difference [F=1.02, P=0.38]. In FIG. 2A, normalization issuggested by a lower score. In FIG. 2B, normalization is indicated bythe opposite direction of change.

MMN and P300 amplitude reflect activation of multiple precortical andcortical pathways sensitive to stimulus novelty, short term memory, andattention. MMN was calculated as the voltage difference over 100-200msec post-stimulus onset between the responses to the rare and frequentstimuli. A more negative MMN suggests normal cognitive function. P300 isnot entirely independent of MMN. P300 was calculated as the peakamplitude relative to the average voltage of the waveform during the 50msec prestimulus period. A more positive P300 response is indicative ofimproved cognitive function. P300 is maximal in amplitude when theeliciting stimulus is both rare and task relevant (i.e., attended). Inthe present study, the rare stimulus was not task relevant. In fact, thepatient was instructed to perform no task and to ignore the stimuli. Inthe present study, therefore, P300 amplitude is very small in comparisonto amplitudes recorded under active task conditions. The present P300component is more similar to the small, frontally-generated P300adescribed by Knight and colleagues than the large, parietally-generatedP300b described in most studies of attentional dysfunction inschizophrenia.

In the analysis of P50 and N100 the baseline value was the covariate andall values obtained during the treatment period were averaged together.Data loss from unidentifiable MMN and P300 components was minimal. Theseanalyses were conducted upon data obtained from n=4 patients treatedwith placebo, n=7 patients treated with 0.3 mg of the test compound, andn=8 patients treated with 1.0 mg of the test compound.

FIGS. 3A and 3B show the results of the analysis of MMN and P300amplitudes during the oddball task. Both evoked potential componentswere sensitive to the test compound in the predicted direction: MMN[F=4.96, P=0.02]; P300 [F=6.88, P=0.008]. In a dose-related manner, thetest compound increased MMN and P300 amplitudes.

Despite the small number of patients enrolled in this trial, theanalysis revealed several significant or marginally significant results.Both the 0.3 mg and 1.0 mg doses of the test compound evokedsignificantly (p<0.05) larger P300 and MMN components than were seenunder the placebo condition. The effects of the test compound on anearlier component of the evoked response component (i.e., the P50) werelimited to the highest, 1.0 mg, dose and were technically notsignificant (p=0.1). These results indicate that both the 0.3 mg doseand 1.0 mg dose of the test compound are anticipated to be effective intreating schizophrenia.

The relative sensitivity or insensitivity of various evoked responsecomponents to the test compound may be related to their size andreliability of measurement. In addition, sensitivity differences mayrelate to differences across the components in their neural generatorsand innervation by cholinergic afferents. Indeed, the two components(MMN and P300) which were most sensitive to the test compound aregenerated or modulated by frontal cortical pathways that receive inputfrom brainstem cholinergic fibers. The P50 is, in contrast, generatedsubcortically.

Effect on Cognition in Normal Subjects

The impact of the test compound on cognition in normal subjects wasassessed as described below. In these studies, subjects were treatedwith the test compound dissolved in cranberry juice.

The impact of the test compound on cognition in normal subjects wasassessed in a SAD (Single Ascending Dose) study with the Digit SymbolSubstitution Test (DSST). Utilizing this test, the test compound wasshown to have pro-cognitive effects at a daily dose as low as 1 mg. Thisis unexpected since acetylcholine esterase inhibitors, which indirectlyactivates the alpha7 receptor by increasing acetylcholine levels, arenot understood to exhibit pro-cognitive effects in normal subject and,even in patients with cognitive impairment, are not understood toexhibit pro-cognitive effects after a single dose. The positive effectsof the test compound in the DSST indicate a beneficial effect on workingmemory and executive function.

In the MAD (Multiple Ascending Dose) studies cognition was assessedusing tests from the CogState battery (cogstate.com). Utilizing thistest, the test compound was shown to have pro-cognitive effects at dailya dose as low as 1 mg. The CogState battery is a proprietarycomputerized cognitive battery of tests measure various cognitivedomains including: attention, identification capability, working memory,visual memory, and executive function. In these studies, the testcompound was found to have a positive impact on: visual motor skills,learning, executive function, and delayed memory. The profile of theresponse was unique insofar as the test compound had positive effects onnon-verbal learning and memory and executive function without having astimulatory effect on attention. The magnitude of the effects were, inmany cases, significant with effect sizes being >0.4 (a threshold effectsize which is commonly accepted as having clinical significance). Thistherapeutic profile (pro-cognitive effects on nonverbal learning andmemory and executive function without a central stimulatory effect)indicates that the drug may be very beneficial in treating patients thathave, as a feature of their condition, symptoms of anxiety or agitation.

(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamidehydrochloride Shows Effects at Unexpectedly Low Dose and Free PlasmaConcentration

The studies described above demonstrate that(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamidehydrochloride administered at a daily dose of 1.0 mg or 0.3 mg canimprove cognition in patients suffering from schizophrenia and in normalsubjects.

The fact that a 0.3 mg or 1.0 mg dose of(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamidehydrochloride can elicit an effect in various measures of cognition issurprising because at these dosages the concentration of free drug iswell below the K_(i) of the compound to bind to the alpha7 receptor.

In order for a small molecule to exert action at its target, often acell receptor, it must bind to its target. Thus, in general, a smallmolecule drug is expected to exhibit activity when the free drugconcentration at the target (i.e., the concentration of drug that isfree and available to bind to the target) approaches or exceeds the K,of the drug for target. Studies have shown that in numerous cases thefree drug concentration in a particular tissue is about equal to thefree drug concentration in plasma (Mauer et al., 2005 and Trainor,2007). For the brain, the free plasma concentration is generallyconsidered to represent the maximum possible free drug concentration.The free drug concentration in plasma ([free drug]_(plasma)) isdetermined by measuring the total drug concentration in the plasma([total drug]_(plasma)) and the free fraction of the drug, i.e., thefraction of the drug that is not bound to plasma protein (fu_(plasma)):[free drug]_(plasma)=[total drug]_(plasma)×fU_(plasma). The total plasmadrug concentration and the fraction that binds to plasma protein canboth be measured using techniques known to those of skill in the art.

Studies on(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamidedetermined that the EC_(ref) for human alpha7 receptor is approximately0.158 μM and the K_(i) (rat membrane) is approximately 10 nM. Additionalstudies found the following values for the free fraction of drug: Ratfu_(plasma)=0.112, Dog fu_(plasma)=0.107, Human fu_(plasma)=0.129.

Multiple ascending dose (MAD) human clinical trials were conducted. Themaximum plasma concentration was determined and used to calculate themaximum free drug concentration which was used to determine the maximumfree drug concentration as a fraction of the EC_(ref) of the drug forhuman alpha7 receptor and the maximum free drug concentration as afraction of the K_(i) of the drug for rat brain alpha7 receptors. TheEC_(ref), the concentration of drug which elicits equal response inoocytes transfected with cloned human alpha7 receptor at 50 μMacetylcholine (the endogenous receptor ligand), was determined to be0.158 μM. The K_(i) for rat brain alpha7 receptors was determined to be10 nM.

TABLE 1 C_(max) C_(max) Fraction Fraction C_(max) total free of α7 of α7Study Day Dose (ng/mL) (nM) (nM) EC_(ref) Binding K_(i) SAD 1 1 mg 0.591.84 0.237 0.0015 0.0237 SAD 1 3.5 mg 2.06 6.42 0.828 0.0052 0.0828 MAD11 1 mg 0.63 1.96 0.252 0.0016 0.0252 MAD1 7 1 mg 2.12 6.61 0.853 0.00540.0853 MAD1 14 1 mg 2.64 8.23 1.06 0.0067 0.1060 MAD2 1 0.1 mg 0.0550.172 0.022 0.0001 0.0022 MAD2 21 0.1 mg 0.232 0.724 0.093 0.0006 0.0093MAD2 1 1 mg 0.623 1.943 0.251 0.0016 0.0251 MAD2 21 1 mg 2.42 7.5470.974 0.0062 0.0974 MAD3 1 0.3 mg 0.182 0.568 0.073 0.0005 0.0073 MAD321 0.3 mg 0.704 2.195 0.283 0.0018 0.0283 MAD3 1 1 mg 0.547 1.71 0.2210.0014 0.0221 MAD3 21 1 mg 1.99 6.20 0.800 0.0051 0.0800

In human single and multiple ascending dose clinical trials in bothhealthy and schizophrenia patients, a 0.3 mg daily dose and a 1.0 mgdaily dose were shown to improve cognitive function or correlates ofcognitive function. As can been seen from Table 1 which presents ananalysis of the free drug concentration, the 0.3 mg dose of(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamidehydrochloride produces a maximum free plasma concentration of0.073-0.283 nM which is 0.005 to 0.0018 of the alpha7 EC_(ref) and0.0073 to 0.0283 of the alpha7 K_(i). These values are 35-2000 timeslower than would have anticipated if efficacy was to be achieved whenthe free plasma concentration reached the K_(i) or the EC_(ref)concentrations. When a similar calculation is performed for the 1.0 mgdoses (free plasma of 0.237-1.06 nM) these fractional values of theK_(i) and EC_(ref) concentrations are 0.0015 to 0.0067 (EC_(ref)) and0.0237 to 0.106 (K_(i)). These values are 9.4-667 times lower thanexpected.

Half-life of(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide inHumans

Table 2 presents half-life (t_(1/2)) data for(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamideobtained from pre-clinical species as well as the half-life in humansdetermined in clinical trials.

TABLE 2 Route of Species Administration Dose t_(1/2) Mouse i.v. n/a* Rati.v. 1 mg/kg 2.77 h Dog i.v. 0.5 mg/kg 5.39 Dog i.v. 3 mg/kg 13 Humanp.o. 1 mg 50.1-70.1*(R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide wasunstable in mouse plasma and as such a half-life (t_(1/2))could not beaccurately calculated.

The half-life determined in rat and dog suggested a human half-life muchshorter than the observed 60 hr half-life (initial allometric scalingsuggested a half-life of about 8 hours). The unexpectedly long half-lifein humans has several advantages. It allows for once a day dosing. Thedrug will also have a very small dynamic plasma range over the course ofa day (about 15-20%). Thus, if a patient misses a daily dose, the plasmalevel and the consequent brain level will not be altered by a greatdegree. This means that the beneficial effects of the drug will be lessdependent upon careful adherence to a specific dosing scheme. Third,long half-life and slow elimination also mean that the final dose willbe lower than expected. This readily observed by looking at the C_(max)values on Day 1 versus Day 21. The C_(max) values on Day 21 are about3.6-4.2 times higher than the Day 1 values. This ratio will translateinto a dose that is 3.6-4.2 times lower than would normally be expecteddue to this favorable accumulation.

REFERENCES

-   Adler, L. E., Hoffer, L. D., Wiser, A., Freedman, R., 1993.    Normalization of auditory physiology by cigarette smoking in    schizophrenic patients. Am J Psychiatry 150, 1856-1861.-   Adler, L. E., Olincy, A., Waldo, M., Harris, J. G., Griffith, J.,    Stevens, K., Flach, K., Nagamoto, H., Bickford, P., Leonard, S.,    Freedman, R., 1998. Schizophrenia, sensory gating, and nicotinic    receptors. Schizophr Bull 24, 189-202.-   Baldeweg, T., Wong, D., Stephan, K. E., 2006. Nicotinic modulation    of human auditory sensory memory: Evidence from mismatch negativity    potentials. Int J Psychophysiol 59, 49-58.-   Boutros, N. N., Overall, J., Zouridakis, G., 1991. Test-retest    reliability of the P50 mid-latency auditory evoked response.    Psychiatry Res 39, 181-192.-   Dalebout, S. D., Fox, L. G., 2001. Reliability of the mismatch    negativity in the responses of individual listeners. J Am Acad    Audiol 12, 245-253.-   de Wilde, O. M., Bour, L. J., Dingemans, P. M., Koelman, J. H.,    Linszen, D. H., 2007. A metaanalysis of P50 studies in patients with    schizophrenia and relatives: differences in methodology between    research groups. Schizophr Res 97, 137-151.-   Dierks, T., Frolich, L., Ihl, R., Maurer, K., 1994. Event-related    potentials and psychopharmacology. Cholinergic modulation of P300.    Pharmacopsychiatry 27, 12-1 A.-   Dunbar, G., Boeijinga, P. H., Demazieres, A., Cisterni, C,    Kuchibhatla, R., Wesnes, K., Luthringer, R., 2007. Effects of    TC-1734 (AZD3480), a selective neuronal nicotinic receptor agonist,    on cognitive performance and the EEG of young healthy male    volunteers. Psychopharmacology (Berl) 191, 919-929.-   Duncan, E., Madonick, S., Chakravorty, S., Parwani, A., Szilagyi,    S., Efferen, T., Gonzenbach, S., Angrist, B., Rotrosen, J., 2001.    Effects of smoking on acoustic startle and prepulse inhibition in    humans. Psychopharmacology (Berl) 156, 266-272.-   Easton, C. J., Bauer, L. O., 1997. Beneficial effects of thiamine on    recognition memory and P300 in abstinent cocaine-dependent patients.    Psychiatry Res 70, 165-174.-   Freedman, R., Leonard, S., Gault, J. M., Hopkins, J., Cloninger, C.    R., Kaufmann, C A., Tsuang, M. T., Farone, S. V., Malaspina, D.,    Svrakic, D. M., Sanders, A., Gejman, P., 2001. Linkage    disequilibrium for schizophrenia at the chromosome 15q13-14 locus of    the alpha7-nicotinic acetylcholine receptor subunit gene (CHRNA7).    Am J Med Genet 105, 20-22.-   Fuerst, D. R., Gallinat, J., Boutros, N. N., 2007. Range of sensory    gating values and test-retest reliability in normal subjects.    Psychophysiology 44, 620-626.-   Heinrichs, R. W., 2004. Meta-analysis and the science of    schizophrenia: variant evidence or evidence of variants? Neurosci    Biobehav Rev 28, 379-394.-   Kaga, K., Harrison, J. B., Butcher, L. L., Woolf, N. J.,    Buchwald, J. S., 1992. Cat ‘P300’ and cholinergic septohippocampal    neurons: depth recordings, lesions, and choline acetyltransferase    immunohistochemistry. Neurosci Res 13, 53-71.-   Katada, E., Sato, K., Sawaki, A., Dohi, Y., Ueda, R., Ojika,    K., 2003. Long-term effects of donepezil on P300 auditory    event-related potentials in patients with Alzheimer's disease. J    Geriatr Psychiatry Neural 16, 39-43.-   Maurer, T., DeBartolo, D., Tess, D., and Scott, D., 2005. The    relationship between the exposure and non-specific binding of    thirty-three central nervous system drugs in mice. Drug Metabolism    and Disposition 33, 175-181.-   Naatanen, R., Alho, K., 1995. Generators of electrical and magnetic    mismatch responses in humans. Brain Topogr 7, 315-320.-   Olincy, A., Harris, J. G., Johnson, L. L., Pender, V., Kongs, S.,    Allensworth, D., Ellis, J., Zerbe, G. O., Leonard, S., Stevens, K.    E., Stevens, J. O., Martin, L., Adler, L. E., Soti, F., Kern, W. R.,    Freedman, R., 2006. Proof-of-concept trial of an alpha7 nicotinic    agonist in schizophrenia. Arch Gen Psychiatry 63, 630-638.-   Potter, D., Summerfelt, A., Gold, J., Buchanan, R. W., 2006. Review    of clinical correlates of P50 sensory gating abnormalities in    patients with schizophrenia. Schizophr Bull 32, 692-700.-   Sandman, C. A., Patterson, J. V., 2000. The auditory event-related    potential is a stable and reliable measure in elderly subjects over    a 3 year period. Clin Neurophysiol 111, 1427-1437.-   Schall, U., Johnston, P., Todd, J., Ward, P. B., Michie, P.    T., 2003. Functional neuroanatomy of auditory mismatch processing:    an event-related fMRI study of duration-deviant oddballs. Neuroimage    20, 729-736.-   Trainor, G, 2007. The importance of plasma protein binding in drug    discovery. Expert Opinion in Drug Discovery 2:51-64.-   Turetsky, B. I., Calkins, M. E., Light, G. A., Olincy, A.,    Radant, A. D., Swerdlow, N. R., 2007. Neurophysiological    endophenotypes of schizophrenia: the viability of selected candidate    measures. Schizophr Bull 33, 69-94.-   Umbricht, D., Krljes, S., 2005. Mismatch negativity in    schizophrenia: a meta-analysis. Schizophr Res 76, 1-23.-   Werber, A. E., Klein, C, Rabey, J. M., 2001. Evaluation of    cholinergic treatment in demented patients by P300 evoked related    potentials. Neural Neurochir Pol 35 Suppl 3, 37-43.

1. A method for improving cognition comprising administering to asubject (R)-7-chloro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamideor a pharmaceutically acceptable salt thereof at a daily dose of lessthan 3 mg.
 2. The method of claim 1 wherein the daily dose is 1 mg orless.
 3. The method of claim 2 wherein the daily dose is 0.3 mg or less.4. (canceled)
 5. The method of claim 1 wherein the daily dose is 1 mg orless.
 6. The method of claim 5 wherein the daily dose is 0.3 mg or less.7. (canceled)
 8. The method of claim 1 wherein the daily dose is 1 mg orless.
 9. The method of claim 8 wherein the daily dose is 0.3 mg or less10. (canceled)
 11. The method of claim 1 wherein the daily dose is 1 mgor less.
 12. The method of claim 11 wherein the daily dose is 0.3 mg orless.
 13. (canceled)
 14. The method of claim 1 wherein the daily dose is1 mg or less.
 15. The method of claim 14 wherein the daily dose is 0.3mg or less.
 16. (canceled)
 17. The method of claim 1 wherein the dailydose is 1 mg or less.
 18. The method of claim 17 wherein the daily doseis 0.3 mg or less.
 19. (canceled)
 20. The method of claim 1 wherein thedaily dose is 1 mg or less.
 21. The method of claim 20 wherein the dailydose is 0.3 mg or less.
 22. (canceled)
 23. The method of claim 1 whereinthe daily dose is 1 mg or less.
 24. The method of claim 11 wherein thedaily dose is 0.3 mg or less.
 25. The method of claim 1, wherein thesubject is suffering from anxiety or agitation. 26-28. (canceled)